EXPERIMENTAL. SCURVYOP THE GUINEA PIG

THE ANTISCORBUTIC VALUE OF MILKAND MILK PRODUCTS

BY

HELEN NKLLORA MINER

THESIS

FOR TBE

DEGREE OF BACHELOR OP SCIENCE

IN

GENERAL SCIENCE

COLLEGE OF LIBERAL ARTS AND SCIENCES

UNIVERSITY OF ILLINOIS

UNIVERSITY OF ILLINOIS

June 14, IS 19 190

THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION BY

_ Helen. Nell ora..Miner

ENTITLED - Experimental Scurvy . of the .....Guinea Pig. The Antiscorbutic Value

of Milk and Milk Products.

IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE

DEGREE OF Bachelor of Science

General Science

Instructor in Charge

APPROVED:

HEAD OF DEPARTMENT OF Chemistry

43395S

•••

iJHJC

TABLE OF CONTENTS

Kasuac of the Disease of Scurvy

Exper imental ScurvyConflicts between vctrious workers

Pro blem

Diagnosis of Experimental Scurvy

Exper lmsntal Work

Series I

Series II

Table s

Charts

Conclusions

Acknowl edgment

Eibliography

Digitized by the Internet Archive

in 2013

http://archive.org/details/experimentalscurOOmine

1

Scurvy is by no means a new disease and altho racent '.vork has brought it be-

fore the aye of the scientific worker we have record of its appearance as far back

at the time of the Crusaders. In de Joinville's account of the Crusade of Louis

XI we are told that scurvy attacked the troops in Palestine. This malady was very

common among the crews of the old sailing vessels which were often at sea for a

period of many months and were frequently deprived of fresh foods. We have a

record of a scurvy epidemic in Dublin in I2H7 and if we turn to the various mili-

tary records we find that scurvy was at one time the scourge of the army. In the

Medical and Surgical History of the War of the Rebellion luring the five and one-

half years covered by the statistics there are reported 30,71^ cases of scurvy

among the white troops with 383 deaths attributed directly to that disease. This

report states further that scurvy developed at practically all of the military

posts during the winter seasons. At posts which could be readily supplied with

potatoes there was only a slight trace of the disease. Hut of still greater in-

terest are the reports of the appearance of scurvy during the recent World War.

It has not only affected the troops but the civilian population as well. Harvier

(l$17) a French surgeon states that S3 percent of the 800 troops of which he had

charge suffered from scurvy and later other epidemics centers were recognized out-

side this sector. Another author reports scurvy among the Italian troops. Germany

likewise suffered from this disease if we can Judge from the condition of the

prisoners of war who were captured in the beginning of 1517. Scurvy is not un-

common in Russia and her troops have felt the effect of this disease during the

past four years. In some parts of the world it would seem that scurvy is still a

Berious problem. It is difficult to estimate,according to Hess, how comcon scurvy

and especially infantile scurvy,, is in the United States. Scurvy is a disease

which requires several months to develop in man and we have no means of knowing

exactly the many nutritional disorders which are in all probability latent cases

of scurvy.

Scurvy has long been recognized as a nutritional disease and practically all

reports of scurvy cases have stated that its appearance was preceded by a monoton-

ous diet which was lacking in fresh foods. When the diet was changed to one of

fresh foods, especially green vegetaoles there was a decided i.ipr overrent in the

condition and recovery was brought about by continuing this diet. Orange juice

and lemon Juice are among, the most potent antiscorbutic foods known. Lime Juice

«vhioh wu soused on the old sailing vessels to alleviate scurvy and in irtic expedi-

tions is believed by recent investigators to have been lemon juice. Chick, Hume,

Skelton and Smith (1$1S) have found that the juice of the lime is only about one-

fourth as effective as that of the lemon ana that the preserved lime juice is

useless. Chick and Rhodes (1918) 3tate the juice of the raw swede (rutabago^ was

found to be practically as effective as the orange juice in averting scurvy symp-

toms. Hess in his work in New York City with both human and experimental scurvy

has pointed out the relationship of the two altho such a relation has been ques-

tioned by some of the other workers. He states that the clinical symptoms are the

same but in many cases tr.ere has been a neglect of microscopic examination of the

bones of the animals ^ffected with scurvy and if this had been done some cases of

animal scurvy would probably have been eliminated as rickets or pseudoscurvy. One'

will recall that the military posts which received a supply of potatoes had only

a "taint" of scurvy. Hess and Unger (I519 )have shown that potato is effective as

a cure when given to tnfanta as well as to guinea pigs.

To Hoist and Frolich (I9O7, 1?12, 1P13 ) we owe the discovery of experimental

•curry in animals. These investigators have shown that by feeding guinea pigs

on a diet composed of cereals they were able to produce a condition which was very

similar to human scurvy. The clinical pictures were very similar except that

"affection of the gums is not usually found in the guinea pig." By the addition

5

of a fresh vegetable to the diet, such as caubage or carrots, the animal was well

nourished and showed no signs of scurvy. Jackson and Moore (I9I0) working with

guinea pigs found that they were able to produce experimental scurvy with various

diets. One series of animals was given a diet of pasturized market milk, oats,

hay and water; another series was given a diet in which the pasturized milk was

replaced by fresh whole milk; and still another series received a diet in which

milk boiled 10 minutes was substituted for the pasturized milk. But in none of

their work was the individual food intake recorded and many of the latter workers

have questioned their results because of this neglect. I.IcCollum and Pitz ( 13 1 7

)

call attention to the peculiar anatomy of the cecum of the guinea pig which they

believe'*coniucivs to constipation and state that "the undue retention of feces is

the primary causes of experimental scurvy in the guinea pig." Scurvy in the

guinea pig. according to these authors, i3 not the result of the deficiency of a

specific protective substance in the food tut rather due to toxic products or

bacterial action. According to Pitz (1313) scurvy is due to the absorption of

putrefactive products of the cecum and anything which will improve or change the

intestinal flora from a putrefactive type should prevent the onset of scurvy. He

found that by adding lactose to a diet of oats and milk scurvy was not only pre-

vented but cured. Here again the amount of milk consumed was not measured and

since the individual intake is so variable it is questionable whether the results

obtained were as accurate as the author would lead us to believe. Harden and

Zilva (151?) U3ed levulose, cane sugar, lactose and the uncrystallized residues

from the preparation of levulose from inulin in their work with guinea pigs but

none of these substances afforded any protection against scurvy. These investi-

gators believe that the results obtained by McCollum and Pitz (191?) and Pitz

(1913) was due to the enhanced consumption of fresh raw milk. Cohen and ."lendel

(I3IS) were unable to effect a cure by the use of lactose and suggest that the

result* Pitz obtained may have been aue to small unsuspected amounts of vitan.ine

which have been shown to be associated with the lactose used in certain nutrition

experiments with isolated food stuffs. Druramond (1316). Those same WOTken say-

that constipation is not responsible for the appearance of scurvy as "cColluui and

Pitz have stated, but thru its complications the severity of the disease nay be

enhanced. They have also found that scurvy can be produced at will by suitably

chosen diets and even tho the diet contains all of the essentials of a well-

balanced ration scurvy develops. These investigators have used milk in their

experiments and the results which they have obtained indicate that milk does con-

tain an antiscorbutic to a small degree.

Chick, Hume, and Skelton (1915) working at the Lister Institute have given

us sots very interesting results concerning the value of milk as an antiscorbutic.

They have found that fresh cow's milk contains an antiscorbutic substance but in

small amounts. Altho the onset of the disease was delayed and the growth of the

animal was maintained in proportion to the daily consump tion of milk a diet con-

sisting almost exclusively of milk was necessary to prevent scurvy. Amounts of

milk ldss than 50 cc. gave very little protection as death from scurvy resulted

in 50 days while 5 3 cc. gave increased protection ana the life of the animal, in

one experiment, was prolonged to 75 days; when the daily intake was increased to

89-130 cc. good health was maintained thruout the period of their experiment which

covered in one case a period of 113 days. The same workers have found that this

antiscorbutic which is present in fresh milk is destroyed by heating in an auto-

clave at 120° for 1 hour, and also by drying and they state that when it is

necessary to use milk in infant feeding which has been heated or dryed an addition-

al antiscorbutic substance, such as orange Juice, should be added, a conanon

pediatric practice in this country.

It seemed desirable to confirm the work of Chick, Hume, and Skelton as to

5

the antiscorbutic value of raw milk gince there wa« a question concerning the

work which Jackson and Moore (1916) and Pitz (1918) had done because of their

failure to measure the amounts of milk actually consumed. In view of the fact

that in their experiments on heated milk these English investigators had used

milk autoclavod at 120° for 1 hour it was Jecided also to extend the study to

milk subjected to somewhat less ri fcorous treatment. Accordingly an investigation

of the antiscorbutic value of milk boiled as is customary in pediatric practice

has been made. It was hoped that some experiments could be carried out upon con-

dented milk and milk powaers, but lack of time gave no opportunity for this fur-

ther study.

Before taking up the experimental work it seems advisable to describe what

is understood to be experimental scurvy of the guinea pig from the clinical stand-

point and also the conditions which the autopsy of a well defined case of scurvy

reveals. Chick, Hume and Skelton (1918) have given the most complete statement

of scurvy symptoms and the observations made in the present work have been ua3ed

upon the symptoms as outlined by these authors. "The first symptoms to be ob-

served is soreness of joints and limbs more especially of shoulders and knees, so

that the animal squeaks when pressure i3 applied to the places. Some animals

squeak when handled under any circumstances and when in perfect health; but if

they are examined regularly from the beginning of the experiment it is possible

to distinguish those which are feeling pain. The presence of painful members is

also shown by the assumption of what we have called the 'scurvy position' which

seems to indicate hemorrhage and consequent discomfort in the muscles of the

limbs. The animal rests on its side and the painful leg is held off the ground

and may be seen twitching. A second attitude which we have called the 1 face-ache

position' is also Indicative of" scurvy in young guinea pig3, the animal lies

curled up with the side of its face pressed on the floor of the cage. This is a

frequent attitude in adult guinea-pigs when in normal health but we have never

teen a young animal adopt it except when ill with scurvy. It seams to indicate

hemorrhage of the Jaw, with soroness and looseness of the teeth. The state of

the molar teeth and of the whole gums cannot he inspected during life and it is

only possible to judge of the condition by the greater or le38 capacity for eat-

ing and by the assumption of the 'face-ache position'." -A t autopsy the most

noticeable condition is the hemorrhagic areas which are found subcutaneously

around the shoulder and knee Joints, and intramuscularly. The costo-chondral

junction is enlarged and hemorrhagic and there is marked fragility of the bones.

There is often a marxed hemorrhage across the sternum and in some few cases there

has been a noticeable loosening of the teeth.

Ixper imental work:

Since various workers have shown conclusively chat experimental scurvy can be

produced in the guinea pig on a cereal diet it was decided to make oatmeal the

foundation of the diet. Oatmeal is not an adequate diet according to ^cColluin,

Simmonds and Pitz ( 15 1 7 ) , since it is lacking in protein and in inorganic salts.

It was thot that in supplementing the oatmeal with milk the protein requirements

would be met by the casein, the inorganic salts would be furnished, and in addi-

tion we would have the two essentials, fat soluble A and water soluble B, which

McCollum says are necessary. In the first series of experiments fresh carrots

which had been boiled for 1 hour were added to the diet to give roughage since

McCollum and Pitz (1917) have stated that a guinea pig can thrive only on a diet

possessing such physical properties a3 will lead to the formation of bulky easily

eliminable fece3.

Series I

Six animals which had been under observation for 10 days were chosen for

the first set of experiments. These ranged in weight from 312 grams to 572 grams

at the beginning of the experiment. They were kept in steel cages with wire

bottoms of small mesh with removable tray beneath. These cages were found much

7

more satisfactory for the work with milk than the cages with the metal bottoms

because of the large volumes of urine which are generally excreted. Guinea pigs

No. 1, 2 ani 7 were given a diet of oatmeal, ad libitum, 35 grams coiled carrots

and boiled milk. The milk in this work was heated to the boiling point and then

removed from the flame. The daily consumption of oats was weighed and the amount

of milk measured altho the animal was, in most cases^ allowed to drink all it cared

for. In this work attempt was ua.de to keep the daily intake of milk up to cc.

or above. In oraer to do this it was often necessary to resort to forced feeding

with a pipette. No. 1 was kept on this diet for 76 days after which the carrots

were removed. On the ?3rd day this animal showed clinical si^rns of 3curvy, it

continued to gain in weight altho it showed every symptom of scurvy. On the 37th

day its condition seemed inproved and by the ^5th day there was no clinical evi-

dence of scurvy. After removing the carrots the animal's weight began to go down •

(Chart I) and on the 30th day scurvy symptoms were again in evidence. It was

killed on the lOUth day because of a severe infection which had developed around

the anu3. It had been necessary to remove him from the wire cage to one with a

steel bottom and because this did not drain well the animal often sat in the

urine. The autopsy showed very marked subcutaneous hemorrhages around the knees

and thru out the thoracic cavity. There were marked intramuscular hemorrhages in

the hind legs and in the thoracic cavity. There was a very marked hemorrhage aero 3

the sternum and the costo-chondral junction was decidedly hemorrhagic. The ends

of the ribs were somewhat enlarged tho there was no enlargement of the joints. The

bones were very fragile and the teeth were slightly loose.

No. 2 was given the diet of oatmeal, boiled milk and boiled carrots for a

period of 60 days. The carrots were then removed from the diet and on the 88th

day scurvy symptoms were first noticed. The consumption of milk had been 5^ cc.

or above after the 20th dav. Up to this time the animal had refused to coorerate

and what milk it got hud to be forced down it, and even then there was much re-

sistance. The animal's feet became very sore and it was thot best to remove it

to a cage with a metal bottom. As in the case of No. 1, a serious infection re-

sulted and the animal was billed on the S^th 'day. There was a rapid loss in

weight after the development of the scurvy symptoms and the general conlition of

the animal was bad. It ahowei loss of appetite ana evacuations were :<j£xie with

great difficulty. Vhe autopsy showed marked scurvy iesiona ('iable I).

Guinea pig No. 7 (Table III) which was given the diet 02' oatmeal, boiled milk

and carrots, showed clinical sumptoms of scurvy on the 2*+th day and these con-

tinued thru out the remaining days of the experiment. It refused to eat the

carrots alter the 28th day. It was killed on the 71st day because it was found

to be infested with lice. Autopsy revealed a well defined case of scurvy.

Guinea pig No. o was used as a control for this series and was given a diet

of oatmeal ana coiled carrots. This animal maintained itself with a slight de-

crease in weight (Chart II) up to the oQth day when the carrots were removed. It

had shown symptoms of scurvy on the lbth day of the experiment but had recovered.

There was a marked decrease in weight following the removal Of the carrots and

on the 6Uth day scurvy symptoms were quite evident. It was killed on the 8Uth day

and the autopsy revealed the typical scurvy picture (Table III).

Guinea pigs Nos. 3 &nd 5 were given the same diet as Nos. 1 and 2 with the

exception that the milk was raw milk. Table II shows the average daily intake

of these animals for each 10-day period of the experiment. No. 3 refused to drink

much milk in the beginning but later became quite greedy for it and consumed 100

80. daily. It developed slight clinical symptoms of scurvy on the 20th day. On

the 28th day the animal had a marked diarrhea which continued until the Ulst day.

On the 31 st day the raw milk was changed to boiled milk with the hops of checking

the diarrhea. The animal was very weak and sore and on the 37th day orange .juice

wag given with the hope of curing him. This was effective and hie condition im-

proved, Chart II, and on the ^5th day the orange juice was discontinue a. After

discontinuing the orange juice, the animal began to fail and lost weight rapidly

tho he was consuming 100 cc. of milk daily. At the beginning he was very eager

for both the oatmeal and carrots but with the increased consume tion of milk his

appetite for these decreased. He died on the 51st day. The autor. sy showed sligh

hemorrhages in the ribs but none in the knee joints. The death of this ani.nal

was probably due to the increased mil* intake (Cohen and .-iendel 1$12) rather than

to scxirvy, since the animal was in such a weakened condition due to the severe

diarrhea.

Guinea pig No. 5 lived for 27 days on the diet of oatmeal, carrots and raw

milk. The milk consumption was low and on the 20th day scurvy symptoms were no-

ticeable. The autopsy revealed a mild case of scurvy.

The boiled carrots which were given to the animals in series I evidently

did not have their antiscorbutic properties destroyed as the animals which re-

ceived 35 grams daily lived a longer period of time than those who received less.

When milk is heated to the boiling point and then removed from the flame its anti

scorbutic value is not changed appreciably. By comparing the weight curves of

Nos. 1 and 2 (Chart 1) with the might curve of No. 6 (Chart II) it would seem

that the addition of milk to the diet of oatmeal and boiled carrots improves the

diet and the animals not only maintain themselves but grow as well.

Series II

With the next group of animals it was considered advisable to subject the

milk to a more uniform treatment than had previously been done. As it was hoped

that the results obtained from this work would be applicable to infant feeding

the milk was heated as is customary in pediatric practice. The usual treatment

in pediatric work is to bring the milk to a' boil as quickly as possible and boil

for three minutes with conetant stirring to prevent the formation of scum acron

the top. A flame was regulated so that the time required to bring the milk to

the boiling point was exactly four minutes, and this v\»s used thru out this work.

The boiled carrots were not given to the animals of this group as it is relieved

that the carrots which are ooiled for 1 hour do not have their antiscorbutic pro-

perties destroyed. This is in accord with the results obtained by Hess and Unger

(ISIS) in which they show that 35 grams of carrots are necessary to afford pro-

tection against scurvy to guinea pig, and if the carrots are fresh toiling for 1

hour has no effect upon their potency.

The diets of the animals in this group were oatmeal and fresh raw milk in one

series and in the secona series milk which had been boiled as outlined above was

substituted for the raw milk. Guinea pigs Not. 10, 11, 13 and 14 were given a

diet of oatmeal ad libitum and fresh raw milk. All of these animals were young

ranging in weight from IS? grams to 278 grams. Table V shows the average daily

intake of oatmeal and milk and the average change in weight (Chart III).

Guinea pigs Nob. 11, 13 ana lU died on the ^Oth, Uath and 30th days, respec-

tively of the experiment. At the autopsy No. 11 showed marked evidence of scurvy-

Clinical symptoms had been in evidence since the 32ni day. .At no time during the

experiment did No. 1-+ show any sijns of scurvy and the autopsy indicated that

death was due to soue other cause rather than scurvy. No. 13 shewed clinical

symptoms of scurvy on the J 4 th day and at the autopsy there was evidence of a mild

case of scurvy. The lungs were badly congested and the livsr was not normal.

Guinea pig No. 10 has been in the laboratory for 102 days and is in good health

and gaining in weight. On the 31st day this animal showed clinical signs of

scurvy which persisted to the ^Sth day (Table V). Tnr milk intake of this animal

has been gradually increased from UO cc. to 75 ec. and it was after the intake

had been increased to 50 cc. that there was a disappearance of scurvy symptoms.

Guinea pigs Nos. 8, $, 16 and 17 were given a diet of oatmeal and boiled

11

milk. No. 8 died on the 33rd day and the autopey revealed marked sclrvy. The

milk intake was kO cc. thru the period of the experiment. No. 9 died on the 55th

lay. There was a constant decrease in weight and altho he drank milk readily he

refused to eat the oatmeal. The milk intake was increased but this resuHed in a

diarrhea, and it is "believed that the amount of milk which he consumed was not

sufficient to maintain life. At one time, 38th day, the knee joints were slightly

scr-5 hut the autopsy did not reveal scurvy.

Guinea pig No. lo was killed at the end of the 37th day because of weakened

condition. The autopsy gave no evidence of scurvy. The milk intake of this ani-

mal had been 40cc. or more thru out the experi-i^nt.

Guinea pig No. 17 has been* in the laboratory for 60 days. It shows clinical

symptoms of scurvy and its general condition is bad altho the animal is not de-

creasing in weight very rapidly.

"When the guinea pig* receives a diet of oatmeal supplemented with milk, either

raw or boiled the life of the animal is prolonged in nearly all cases. In com-

paring the results of this series with the results of series I there is not the

difference in the effect of the boiled milk as one might expect. The animal must

receive an average daily intake of more than kO cc. of milk in order to have any

marked effect in preventing the onset of scurvy. (No». 8, 16 and 11) No. 10 has

continued to gain in weight steadily altho the clinical symptoms of scurvy were

very evident. The toiled carrots which were fed in series I prolonged the life

of the animals for a longer period of time than does milk alone unless taken in

large quantities.

CONCLUSIONS

Altho it has been impossible to make a complete study of this problem the

following conclusions have been made from the experimental data.

1. Carrots which are coiled one hour do not have their antiscorbutic value

12

entirely destroyed.

2. The consumption of large quantities of milk by guinea pigs may cause

m.-rked intestinal distur bances resulting in severe diarrhea.

3. There is a decided variation among the animals in the consumption of Bilk

and the results which Jackson and Moore (1916 ) and Pitz (1918) have obtained are

probably due to their failure to recognize this f^ct.

4. naw milk has an antiscorbutic property which is present to a small decree

and which is destroyed somewhat by heating as is customary in pediatric practice.

5- Poilei milk, as well as raw milk, does inprove the diet of oatueal and the

life of the guinea pig is prolonged and the appearance of scurvy symptoms delayed

when an intake of UO cc. or more of milk is consumed.

6. Guinea pigs may increase in weight very markedly while showing mild clin-

gs iM^xa^ical scurvy. The development of scurvy clinically is almost invariably accom-

panied by marked loss in weight.

7< From this work it seems advisable to recommend that an antiscorbutic food

shoviid be included in the diet of infants who are given milk which has been sub-

jected to heating.

I wish to acknowledge my indebtedness to Dr. Howard B. Lewis. under whose

supervision this work has been done, and whose frequent suggestions have made

possible the comrletion of the work.

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Oat meal

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gms. gms. gms. laye

1X ^12J J* c 500 361 104 Scurvy

2 706 402 96 Scurvy

* • 7 j *> j 401 319 71 Scurvy

Oatmeal, raw milk and "boiled carrot s

3 572 602 402 51 Mild scurvy Severe diarrhea causedweakened condition.

J ^S4 357 250 27 i»lild 9curvy

h 4b5 512 350 84 Scurvy

Oa tmeal and boiled milk

g 220 227 *7 33 Scurvy

9 276 283 150 55 No scurvy

16 30° 344 215 3711 it

17 355 374 258 60+ Scurvy Animal still alive thosymptoms. shows clinical scurvy.

Oatmeal and raw milk

10 19c 404 404 114+ normal Animal still alive. Seeminglylias fully recovered from

11 189 223 209 39 Scurvy scurvy symptoms noticable on

44 Scurvy (?)

34th day.13 278 282 203 Pneumonia was the real causs

of death tho some scurvylesions visible.

14 277 302 250 40 No 3curvy Killed because 01 weakenedcondition.

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BIBLIOGRAPHY

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Skelton, R. F. , 1918. Biochem. Jour., xii, 131.

Chick, H., Hume, E. M., Skelton, R. F., and Smith, A. H.,

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